Gibberellin 2-oxidase

ABSTRACT

A nucleic acid sequence is provided which encodes a gibberellin 2-oxidase gene which catalyses the 2β-oxidation of a gibberellin molecule to introduce a hydroxyl group at C-2 and further catalyses the oxidation of the hydroxyl group introduced at C-2 to yield the ketone derivative. Such sequences can find application in the preparation of transgenic plants with altered levels of gibberellin 2-oxidase.

CROSS REFERENCE TO RELATED APPLICATIONS

The present application is the U.S. National Phase of InternationalApplication No. PCT/GB9901857, International Application filing dateJun. 11, 1999, which was published in English on Dec. 23, 1999 asWO99/66029, and claims priority under 35 U.S.C. § 119 to United Kingdomapplications GB 9812821.8, filed Jun. 12, 1998, and GB 9815404.0, filedJul. 15, 1998.

BACKGROUND OF THE INVENTION

1. Field of the Invention

The present invention relates to a novel enzyme involved in the controlof plant growth, DNA sequences coding for the enzyme and uses of thenucleotide sequence coding for the enzyme in the production oftransgenic plants with improved or altered growth characteristics.

2. Related Art

The gibberellins (GAs) are a large group of diterpenoid carboxylic acidsthat are present in all higher plants and some fungi. Certain members ofthe group function as plant hormones and are involved in manydevelopmental processes, including seed germination, stem extension,leaf expansion, flower initiation and development, and growth of theseeds and fruit. The biologically active GAs are usually C₁₉ compoundscontaining a 19-10 lactone, a C-7 carboxylic acid and a 3β-hydroxylgroup. The later stages of their biosynthesis involve the oxidativeremoval of C-20 and hydroxylation at C-3. Hydroxylation at the 2βposition results in the production of biologically inactive products.This reaction is the most important route for GA metabolism in plantsand ensures that the active hormones do not accumulate in plant tissues.The GA biosynthetic enzymes 7-oxidase, 20-oxidase, 3β-hydroxylase and2β-hydroxylase are all 2-oxoglutarate-dependent dioxygenases. These area large group of enzymes for which 2-oxoglutarate is a co-substrate thatis decarboxylated to succinate as part of the reaction (see review byHedden, P. and Kamiya, Y., in Annu. Rev. Plant Physiol. Plant Mol. Biol.48 431-460 (1997)).

Chemical regulators of plant growth have been used in horticulture andagriculture for many years. Many of these compounds function by changingthe GA concentration in plant tissues. For example, growth retardantsinhibit the activity of enzymes involved in GA biosynthesis and therebyreduce the GA content. Such chemicals are used commonly, for example, toprevent lodging in cereals and to control the growth of ornamental andhorticultural plants. Conversely, GAs may be applied to plants, such asin the application of GA₃ to seedless grapes to improve the size andshape of the berry, and to barley grain to improve malt production.Mixtures of GA₄ and GA₇ are applied to apples to improve fruit qualityand to certain conifers to stimulate cone production. There are severalproblems associated with the use of growth regulators. Some of thegrowth retardants are highly persistent in the soil making it difficultto grow other crops following a treated crop. Others require repeatedapplications to maintain the required effect. It is difficult torestrict application to the target plant organs without it spreading toother organs or plants and having undesirable effects. Precise targetingof the growth-regulator application can be very labour intensive. Anon-chemical option for controlling plant morphology is, thus, highlydesirable.

Developing seeds often contain high concentrations of GAs and relativelylarge amounts of GA-biosynthetic enzymes. Mature seeds of runner bean(Phaseolus coccineus) contain extremely high concentrations of the2β-hydroxy GA, GA₈, as its glucoside, indicating that high levels of2β-hydroxylase activity must be present. This has been confirmed for therelated species Phaseolus vulgaris in which there is a rapid increase inGA 2β-hydroxylase activity shortly before seeds reach full maturity(Albone et al., Planta 177 108-115 (1989)). 2β-Hydroxylases have beenpartially purified from the cotyledons of Pisum sativum (Smith, V. A.and MacMillan, J., Planta 167 9-18 (1983)) and Phaseolus vulgaris(Griggs et al Phytochemistry 30 2507-2512 (1991) and Smith, V. A. andMacMillan, J., J. Plant Growth Regul. 2 251-264 (1984)). These studiesshowed that there was evidence that, for both sources, at least twoenzymes with different substrate specificities are present. Twoactivities from cotyledons of imbibed P. vulgaris were separable bycation-exchange chromatography and gel-filtration. The major activity,corresponding to an enzyme of M_(r) 26,000 by size exclusion HPLC,hydroxylated GA₁ and GA₄ in preference to GA₉ and GA₂₀, while GA₉ wasthe preferred substrate for the second enzyme (M_(r) 42,000). However,attempts to purify the enzyme activity to obtain N-terminal informationfor amino acid sequencing have proved impossible because of the lowabundance of the enzyme in the plant tissues relative to other proteinsand the co-purification of a contaminating lectin with the enzymeactivity rendering N-terminal amino acid sequencing impossible.

The regulation of gibberellin deactivation has been examined in Pisumsativum (garden pea) using the sln (slender) mutation as reported inRoss et al (The Plant Journal 7 (3) 513-523 (1995)). The sln mutationblocks the deactivation of GA₂₀ which is the precursor of the bioactiveGA₁. The results of these studies indicated that the sln gene may be aregulatory gene controlling the expression of two separate structuralgenes involved in GA deactivation, namely the oxidation of GA₂₀ to GA₂₉by 2β-hydroxylation at C-2 followed by the further oxidation of thehydroxyl group to a ketone (GA₂₉ to GA₂₉-catabolite). The conversion ofGA₂₉ to GA₂₉-catabolite in pea seeds was inhibited byprohexadione-calcium, an inhibitor of 2-oxoglutarate-dependentdioxygenases (Nakayama et al Plant Cell Physiol. 31 1183-1190 (1990)),indicating that the reaction was catalysed by an enzyme of this type.Although the slender (sln) mutation in peas was found to block both theconversion of GA₂₀ to GA₂₉ and of GA₂₉ to GA₂₉-catabolite in seeds, theinability of unlabeled GA₂₀ to inhibit oxidation of radiolabelled GA₂₉,and vice versa, indicated that the steps were catalysed by separateenzymes. Furthermore, in shoot tissues, the slender mutation inhibitsthe 2β-hydroxylation of GA₂₀, but not the formation of GA₂₉-catabolite.These observations lead to the theory that there were two separateenzymes involved in this metabolic pathway controlling the deactivationof GA in plants (Hedden, P. and Kamiya, Y., in Annu. Rev. Plant Physiol.Plant Mol. Biol. 48 431460 (1997)).

BRIEF SUMMARY OF THE INVENTION

However, it has now surprisingly been found that a single enzyme can, infact, catabolise these different reactions. The present inventionrepresents the first reported cloning of a cDNA encoding a GA2β-hydroxylase that acts on C₁₉-GAs and for which 2β-hydroxylation isits only hydroxylase activity. A cDNA clone from pumpkin seed encodes anenzyme that has both 2β- and 3β-hydroxylase activities (Lange et al.Plant Cell 9 1459-1467 (1997)), but its major activity is3β-hydroxylation and it acts as a 2β-hydroxylase only with tricarboxylicacid (C₂₀) substrates; it does not 2β-hydroxylate C₁₉-GAs. Since the newenzyme of the present invention catalyses both the β-hydroxylation andfurther oxidation of the substituted hydroxyl group to a ketone group atC-2, the enzyme has been termed a “GA 2-oxidase”.

DETAILED DESCRIPTION OF THE INVENTION

According to a first aspect of the present invention there is providedan isolated, purified or recombinant nucleic acid sequence encoding agibberellin 2-oxidase enzyme comprising a nucleic acid sequence as shownin FIG. 1 or a functional derivative thereof, or its complementarystrand or a homologous sequence thereto.

A system of nomenclature for the GA-biosynthesis genes has now beenintroduced (Coles et al The Plant Journal 17(5) 547-556 (1999).References in the present application to the gibberellin 2-oxidase geneof Phaseolus coccineus should be understood as also referring toPcGA2ox1. References in the present application to the gibberellin2-oxidase genes of Arabidopsis thaliana as at-2bt3, at-2bt24 andT31E10.11 should be understood as also referring to AtGA2ox1, AtGA2ox2and AtGA2ox3 respectively.

Nucleic acid sequences of the present invention which encode agibberellin 2-oxidase (GA 2-oxidase) are 2-oxoglutarate-dependentdioxygenases that introduce a hydroxyl group at C-2β on GAs,particularly C₁₉-GAs, including the bioactive GAs such as GA₁ and GA₄.They may also oxidise the 2β-hydroxylated GAs further to giveGA-catabolites, which have a ketone function at C-2. The lactone bridgeof these catabolites may also be opened to produce a C-19 carboxylicacid and a double bond at C-10. The activity of the 2-oxidases resultsin inactivation of bioactive GAs or in the conversion of biosyntheticprecursors of active GAs to products that cannot be converted tobioactive forms. A preferred nucleic acid sequence of the presentinvention therefore encodes a gibberellin 2-oxidase enzyme capable ofoxidising C₁₉-gibberellin compounds by introduction of a hydroxyl groupat C-2β. The enzyme may also oxidise the 2β-hydroxyl group to a ketonegroup. Preferred substrates of gibberellin 2-oxidases of the presentinvention are GA₉, GA₄, GA₂₀ and GA₁.

In the context of the present invention, the degree of identity betweenamino acid sequences may be at least 40%, suitably 50% or higher, e.g.55%, 60%, 65%, 70%, 75%, 80%, 85%, 90% or 95%. At the nucleotide level,the degree of identity may be at least 50%, suitably 60% or higher, e.g.65%, 70%, 75%, 80%, 85%, 90% or 95%. A homologous sequence according tothe present invention may therefore have a sequence identity asdescribed above. Sequence homology may be determined using anyconveniently available protocol, for example using Clustal X™ from theUniversity of Strasbourg and the tables of identities produced usingGenedoc™ (Karl B. Nicholas).

Also included within the scope of the present invention are nucleic acidsequences which hybridises to a sequence in accordance with the firstaspect of the invention under stringent conditions, or a nucleic acidsequence which is homologous to or would hybridise under stringentconditions to such a sequence but for the degeneracy of the geneticcode, or an oligonucleotide sequence specific for any such sequence.

Stringent conditions of hybridisation may be characterised by low saltconcentrations or high temperature conditions. For example, highlystringent conditions can be defined as being hybridisation to DNA boundto a solid support in 0.5M NaHPO₄, 7% sodium dodecyl sulfate (SDS), 1 mMEDTA at 65° C., and washing in 0.1×SSC/0.1% SDS at 68° C. (Ausubel et aleds. “Current Protocols in Molecular Biology” 1, page 2.10.3, publishedby Green Publishing Associates, Inc. and John Wiley & Sons, Inc., NewYork, (1989)). In some circumstances less stringent conditions may berequired. As used in the present application, moderately stringentconditions can be defined as comprising washing in 0.2×SSC/0.1% SDS at42° C. (Ausubel et al (1989) supra). Hybridisation can also be made morestringent by the addition of increasing amounts of formamide todestabilise the hybrid nucleic acid duplex. Thus particularhybridisation conditions can readily be manipulated, and will generallybe selected according to the desired results. In general, convenienthybridisation temperatures in the presence of 50% formamide are 42° C.for a probe which is 95 to 100% homologous to the target DNA, 37° C. for90 to 95% homology, and 32° C. for 70 to 90% homology.

An example of a preferred nucleic acid sequence of the present inventionis one which encodes an enzyme which has the activity of a gibberellin2-oxidase enzyme of Phaseolus coccineus (for example PcGA2ox1) or anequivalent protein of another member of the Fabaceae family. A nucleicacid sequence of the present invention may also encode a gibberellin2-oxidase enzyme from Phaseolus vulgaris or from Arabidopsis thaliana(for example AtGA2ox1, AtGA2ox2 or AtGAox3).

Other nucleic acid sequences in accordance with this aspect of thepresent invention may also comprise a nucleic acid sequence aspreviously defined in which the coding sequence is operatively linked toa promoter. The promoter may be constitutive and/or specific forexpression in a particular plant cell or tissue, for example in rootsusing tobacco RB7 (Yamamoto et al Plant Cell 3 371-382 (1991)); in greentissues using tomato rbcS-3A (Ueda et al Plant Cell 1 217-227 (1989));in dividing cells using maize histone H3 (Brignon et al Plant Mol. Biol.22 1007-1015 (1993)), or Arabidopsis CYC07 (Ito et al Plant Mol. Biol.24 863-878 (1994)); in vegetable meristem using Arabidopsis KNAT1(Lincoln et al Plant Cell 6 1859-1876 (1994)); in vascular tissue usingbean GRP1.8 (Keller, B., & Heierli, D., Plant Mol. Biol. 26 747-756(1994)); in flower using Arabidopsis ACT11 (Huang et al Plant Mol. Biol.33 125-139 (1997)) or petunia chalcone synthase (Vandermeer et al PlantMol. Biol. 15 95-109 (1990)); in pistil using potato SK2 (Ficker et alPlant Mol. Biol. 35 425-431 (1997); in anther using Brassica TA29(Deblock, M., & Debrouwer, D., Planta 189 218-225 (1993)); in fruitusing tomato polygalacturonase (Bird et al Plant Mol. Biol. 11 651-662(1988)). Alternative promoters may be derived from plant viruses, forexample the Cauliflower mosaic virus 35S promoter (CaMV). Suitablepromoter sequences can include promoter sequences from plant species,for example from the family Brassicaceae.

The present invention therefore also extends to an isolated, purified orrecombinant nucleic acid sequence comprising a promoter which naturallydrives expression of a gene encoding a gibberellin 2-oxidase enzymecomprising a nucleic acid sequence as shown in FIG. 1 or a functionalderivative thereof, or its complementary strand, or a sequencehomologous thereto. The gibberellin 2-oxidase enzyme may be of Phaseoluscoccineus (for example PcGA2ox1) or an equivalent protein of anothermember of the Fabaceae family. Such nucleic acid sequences may alsoencode a gibberellin 2-oxidase enzyme from P. vulgaris or A. thaliana(for example AtGA2ox1, AtGA2ox2 or AtGA2ox3). Preferably, the nucleicacid sequence comprises a promoter which drives expression of agibberellin 2-oxidase enzyme from P. coccineus, P. vulgaris or A.thaliana. Such promoter sequences include promoters which occurnaturally 5′ to the coding sequence of the sequence shown in FIG. 1.Promoters may also be selected to constitutively overexpress the nucleicacid coding for the gibberellin 2-oxidase gene. Promoters that areinduced by internal or external factors, such as chemicals, planthormones, light or stress could be used. Examples are the pathogenesisrelated genes inducible by salicylic acid, copper-controllable geneexpression (Mett et al Proc. Nat'l. Acad. Sci. USA 90 4567-4571 (1993))and tetracycline-regulated gene expression (Gatz et al Plant Journal 2397-404 (1992)). Examples of gibberellin-inducible genes are γ-TIP(Phillips, A. L., & Huttly, A. K., Plant Mol. Biol. 24 603-615 (1994))and GAST (Jacobsen, S. E., & Olszewski, N. E., Planta 198 78-86 (1996)).Gibberellin 20-oxidase genes are down-regulated by GA (Phillips et alPlant Physiol. 108 1049-1057. (1995)) and their promoter coupled to theGA 2-oxidase ORF may also find application in this aspect of theinvention.

Gibberellin 2-oxidase enzymes coded for by nucleic acid sequences of thepresent invention may suitably act to catalyse the 2β-oxidation of aC₁₉-gibberellin molecule to introduce a hydroxyl group at C-2 followedby further oxidation to yield the ketone derivative.

The nucleic acid sequences of the present invention may also code forRNA which is antisense to the RNA normally found in a plant cell or maycode for RNA which is capable of cleavage of RNA normally found in aplant cell. Accordingly, the present invention also provides a nucleicacid sequence encoding a ribozyme capable of specific cleavage of RNAencoded by a gibberellin 2-oxidase gene. Such ribozyme-encoding DNAwould generally be useful in inhibiting the deactivation ofgibberellins, particularly C₁₉-GAs.

Nucleic acid sequences in accordance with the present invention mayfurther comprise 5′ signal sequences to direct expression of theexpressed protein product. Such signal sequences may also includeprotein targeting sequences which can direct an expressed protein to aparticular location inside or outside of a host cell expressing such anucleic acid sequence. Alternatively, the nucleic acid sequence may alsocomprise a 3′ signal such as a polyadenylation signal or otherregulatory signal.

The present invention therefore offers significant advantages toagriculture in the provision of nucleic acid sequences to regulate themetabolism of the gibberellin plant hormones. The regulation could be toeither inhibit plant growth by promoting the action of gibberellin2-oxidase or to promote plant growth by preventing the deactivation ofgibberellin by gibberellin 2-oxidase. For example, in 1997, there waslodging in about 15% of the wheat and 30% of the barley crop in the UKwith an estimated cost to the growers of £100 m. The availability oflodging-resistant cereals with shorter, stronger stems as a result ofreduced GA content could be of considerable financial benefit.

According to another aspect of the present invention there is providedan antisense nucleic acid sequence which includes a transcribable strandof DNA complementary to at least part of the strand of DNA that isnaturally transcribed in a gene encoding a gibberellin 2-oxidase enzyme,such as the gibberellin 2-oxidase enzymes from P. coccineus, P. vulgarisor A. thaliana. Preferred genes according to the present inventioninclude PcGA2ox1, AtGA2ox1, AtGA2Ox2 and AtGA2Ox3.

The antisense nucleic acid and ribozyme-encoding nucleic acid describedabove are examples of a more general principle: according to a furtheraspect of the invention there is provided DNA which causes (for exampleby its expression) selective disruption of the proper expression ofgibberellin 2-oxidase genes, or in preferred embodiments the P.coccineus gene PcGA2ox1.

According to another aspect of the present invention there is providedan isolated, purified or recombinant polypeptide comprising agibberellin 2-oxidase enzyme having the amino acid sequence as shown inFIG. 2.

Recombinant DNA in accordance with the invention may be in the form of avector. The vector may for example be a plasmid, cosmid, phage orartificial chromosome. Vectors will frequently include one or moreselectable markers to enable selection of cells transfected (ortransformed: the terms are used interchangeably in this specification)with them and, preferably, to enable selection of cells harbouringvectors incorporating heterologous DNA. Appropriate “start” and “stop”signals will generally be present. Additionally, if the vector isintended for expression, sufficient regulatory sequences to driveexpression will be present; however, DNA in accordance with theinvention will generally be expressed in plant cells, and so microbialhost expression would not be among the primary objectives of theinvention, although it is not ruled out (such as for example inbacterial or yeast host cells). Vectors not including regulatorysequences are useful as cloning vectors.

Cloning vectors can be introduced into E. coli or another suitable hostwhich facilitates their manipulation. According to another aspect of theinvention, there is therefore provided a host cell transfected ortransformed with a nucleic acid sequence as described above. A furtherembodiment of the invention is the provision of enzymes by expression ofGA 2-oxidase cDNAs in heterologous hosts, such as Escherichia coli,yeasts including strains of Saccharomyces cerevisiae, or insect cellsinfected with a baculovirus containing recombinant DNA. The enzymescould be used for the production of 2β-hydroxylated GAs andGA-catabolites or for the preparation of antibodies raised against GA2-oxidases. The host cell may also suitably be a plant cell in plantcell culture or as part of a callus.

Nucleic acid sequences in accordance with this invention may be preparedby any convenient method involving coupling together successivenucleotides, and/or ligating oligo- and/or poly-nucleotides, includingcell-free in vitro processes, but recombinant DNA technology forms themethod of choice.

Ultimately, nucleic acid sequences in accordance with the presentinvention will be introduced into plant cells by any suitable means.According to a still further aspect of the invention, there is provideda plant cell including a nucleic acid sequence in accordance with theinvention as described above.

Preferably, nucleic acid sequences of the present invention areintroduced into plant cells by transformation using the binary vectorpLARS120, a modified version of pGPTV-Kan (Becker et al Plant Mol. Biol.20 1195-1197 (1992)) in which the β-glucuronidase reporter gene isreplaced by the Cauliflower mosaic virus 35S promoter from pBI220(Jefferson, R. A., Plant Mol. Biol. Rep. 5 387405 (1987)). Such plasmidsmay be then introduced into Agrobacterium tumefaciens by electroporationand can then be transferred into the host cell via a vacuum filtrationprocedure. Alternatively, transformation may be achieved using adisarmed Ti-plasmid vector and carried by Agrobacterium by proceduresknown in the art, for example as described in EP-A-0116718 andEP-A-0270822. Where Agrobacterium is ineffective, the foreign DNA couldbe introduced directly into plant cells using an electrical dischargeapparatus alone, such as for example in the transformation ofmonocotyledonous plants. Any other method that provides for the stableincorporation of the nucleic acid sequence within the nuclear DNA ormitochondrial DNA of any plant cell would also be suitable. Thisincludes species of plant which are not yet capable of genetictransformation.

Preferably, nucleic acid sequences in accordance with the invention forintroduction into host cells also contain a second chimeric gene (or“marker” gene) that enables a transformed plant containing the foreignDNA to be easily distinguished from other plants that do not contain theforeign DNA. Examples of such a marker gene include antibioticresistance (Herrera-Estrella et al EMBO J. 2 987-995 (1983)), herbicideresistance (EP-A-0242246) and glucuronidase (GUS) expression(EP-A-0344029). Expression of the marker gene is preferably controlledby a second promoter which allows expression in cells at all stages ofdevelopment so that the presence of the marker gene can be determined atall stages of regeneration of the plant.

A whole plant can be regenerated from a single transformed plant cell,and the invention therefore provides transgenic plants (or parts ofthem, such as propagating material, i.e. protoplasts, cells, calli,tissues, organs, seeds, embryos, ovules, zygotes, tubers, roots, etc.)including nucleic acid sequences in accordance with the invention asdescribed above. In the context of the present invention, it should benoted that the term “transgenic” should not be taken to be limited inreferring to an organism as defined above containing in their germ lineone or more genes from another species, although many such organismswill contain such a gene or genes. Rather, the term refers more broadlyto any organism whose germ line has been the subject of technicalintervention by recombinant DNA technology. So, for example, an organismin whose germ line an endogenous gene has been deleted, duplicated,activated or modified is a transgenic organism for the purposes of thisinvention as much as an organism to whose germ line an exogenous DNAsequence has been added.

Preferred species of plants include but are not limited tomonocotyledonous plants including seed and the progeny or propagulesthereof, for example Lolium, Zea, Triticum, Sorghum, Triticale,Saccharun, Bromus, Oryzae, Avena, Hordeum, Secale and Setaria.Especially useful transgenic plants are maize, wheat, barley plants andseed thereof. Dicotyledenous plants are also within the scope of thepresent invention and preferred transgenic plants include but are notlimited to the species Fabaceae, Solanum, Brassicaceae, especiallypotatoes, beans, cabbages, forest trees, roses, clematis, oilseed rape,sunflower, chrysanthemum, poinsettia and antirrhinum (snapdragon).

Screening of plant cells, tissue and plants for the presence of specificDNA sequences may be performed by Southern analysis as described inSambrook et al (Molecular Cloning: A Laboratory Manual, Second edition(1989)). This screening may also be performed using the Polymerase ChainReaction (PCR) by techniques well known in the art.

Transformation of plant cells includes separating transformed cells fromthose that have not been transformed. One convenient method for suchseparation or selection is to incorporate into the material to beinserted into the transformed cell a gene for a selection marker. As aresult only those cells which have been successfully transformed willcontain the marker gene. The translation product of the marker gene willthen confer a phenotypic trait that will make selection possible.Usually, the phenotypic trait is the ability to survive in the presenceof some chemical agent, such as an antibiotic, e.g. kanamycin, G418,paromomycin, etc, which is placed in a selection media. Some examples ofgenes that confer antibiotic resistance, include for example, thosecoding for neomycin phosphotransferase kanamycin resistance (Velten etal EMBO J. 3 2723-2730 (1984)), hygromycin resistance (van den Elzen etal Plant Mol. Biol. 5 299-392 (1985)), the kanamycin resistance (NPT II)gene derived from Tn5 (Bevan et al Nature 304 184-187 (1983); McBride etal Plant Mol. Biol. 14 (1990)) and chloramphenicol acetyltransferase.The PAT gene described in Thompson et al (EMBO J. 6 2519-2523 (1987))may be used to confer herbicide resistance.

An example of a gene useful primarily as a screenable marker in tissueculture for identification of plant cells containing geneticallyengineered vectors is a gene that encodes an enzyme producing achromogenic product. One example is the gene coding for production ofβ-glucuronidase (GUS). This enzyme is widely used and its preparationand use is described in Jefferson (Plant Mol. Biol. Reporter 5 387-405(1987)).

Once the transformed plant cells have been cultured on the selectionmedia, surviving cells are selected for further study and manipulation.Selection methods and materials are well known to those of skill in theart, allowing one to choose surviving cells with a high degree ofpredictability that the chosen cells will have been successfullytransformed with exogenous DNA.

After transformation of the plant cell or plant using, for example, theAgrobacterium Ti-plasmid, those plant cells or plants transformed by theTi-plasmid so that the enzyme is expressed, can be selected by anappropriate phenotypic marker. These phenotypic markers include, but arenot limited to, antibiotic resistance. Other phenotypic markers areknown in the art and may be used in this invention.

Positive clones are regenerated following procedures well-known in theart. Subsequently transformed plants are evaluated for the presence ofthe desired properties and/or the extent to which the desired propertiesare expressed. A first evaluation may include, for example, the level ofbacterial/fungal resistance of the transformed plants, stableheritability of the desired properties, field trials and the like.

By way of illustration and summary, the following scheme sets out atypical process by which transgenic plant material, including wholeplants, may be prepared. The process can be regarded as involving fivesteps:

(1) first isolating from a suitable source or synthesising by means ofknown processes a DNA sequence encoding a protein exhibiting GA2-oxidase activity;

(2) operably linking the said DNA sequence in a 5′ to 3′ direction toplant expression sequences as defined hereinbefore;

(3) transforming the construct of step (2) into plant material by meansof known processes and expressing it therein;

(4) screening of the plant material treated according to step (3) forthe presence of a DNA sequence encoding a protein exhibiting gibberellin2-oxidase activity: and

(5) optionally regenerating the plant material transformed according tostep (3) to a whole plant.

The present invention thus also comprises transgenic plants and thesexual and/or asexual progeny thereof, which have been transformed witha recombinant DNA sequence according to the invention. The regenerationof the plant can proceed by any known convenient method from suitablepropagating material either prepared as described above or derived fromsuch material.

The expression “asexual or sexual progeny of transgenic plants” includesby definition according to the invention all mutants and variantsobtainable by means of known process, such as for example cell fusion ormutant selection and which still exhibit the characteristic propertiesof the initial transformed plant, together with all crossing and fusionproducts of the transformed plant material.

Another object of the invention concerns the proliferation material oftransgenic plants. The proliferation material of transgenic plants isdefined relative to the invention as any plant material that may bepropagated sexually in vivo or in vitro. Particularly preferred withinthe scope of the present invention are protoplasts, cells, calli,tissues, organs, seeds, embryos, egg cells, zygotes, together with anyother propagating material obtained from transgenic plants.

A further aspect of the invention is the provision of an antibody raisedagainst at least a part of the amino acid sequence of gibberellin2-oxidase. Such antibody is useful in screening a cDNA library insuitable vectors derived from the plant tissue RNA.

The gibberellin 2-oxidase gene according to the invention is useful inthe modification of growth and developmental processes in transgenicplants. Another important aspect of the present invention is thereforeits use in the preparation of transgenic plants or seeds in which thegibbereuin 2-oxidase is constitutively overexpressed to reduce theconcentration of bioactive gibberellins (GAs) in the plants or seeds.Preferred gibberellin 2-oxidase genes include PcGA2ox1, AtGA2ox1,AtGA2ox2 and AtGA2Ox3. Such transgenic plants overexpressing the GA2-oxidase would resemble plants that had been treated with growthretardants. The invention could therefore be used to reduce vegetativegrowth as in, for example, the prevention of lodging in cereals,including rice, and the improvement in grain yield, the prevention oflodging in oilseed rape and the improvement of canopy structure, theimprovement in seedling quality for transplantation, the reduction ingrowth of amenity grasses, the reduction in shoot growth in orchard andornamental trees, the production of ornamental plants with more compactgrowth habit, the improvement in tolerance to cold, draught andinfection, the increase in yields by diversion of assimilates fromvegetative to reproductive organs, the prevention of bolting in rosetteplants, such as sugar beet, lettuce, brassicas and spinach. Theinvention may also be used to induce male and/or female sterility byexpression in floral organs, to prevent pre-harvest sprouting incereals, to reduce shoot growth in hedging plants, to inhibit reversiblythe development or germination of seeds and to reduce shoot growth ofcommercial wood species.

Overexpression of the nucleic acid sequences encoding gibberellin2-oxidase may be achieved using DNA constructs comprising constitutivepromoters and nucleic acid coding sequences in transgenic plantsprepared by recombinant DNA technology. Alternatively, theoverexpression may be achieved using the technique of homologousrecombination to insert into the nucleus of a cell a constitutivepromoter upstream of a normally silent copy of the nucleic acid sequenceof the present invention.

The present invention also provides in an additional aspect the use of anucleic acid sequence as previously defined in the preparation oftransgenic plants and/or seeds in which expression of endogenous GA2-oxidase genes in transgenic plants is reduced (i.e. silenced), by, forexample, the expression of antisense copies of the endogenous GA2-oxidase DNA sequences, the expression of truncated sense copies of theendogenous gene (co-suppression) or the use of synthetic ribozymestargeted to the endogenous transcripts. Preferred gibberellin 2-oxidasegenes according to this aspect of the invention include PcGA2ox1,AtGA2ox1, AtGA2ox2 and AtGA20x3.

This would result in plants with reduced turnover, and hence increasedconcentrations, of bioactive GAs. In this form, the invention could beused, for example, to improve fruit set and growth in seedless grapes,citrus and pear, improve skin texture and fruit shape in apple, increasestem length and therefore yield in sugar cane, increase yield andearliness in celery and rhubarb, improve malting yields and quality incereals, particularly barley. It could also be used to increase growthin woody species.

Preferred features of the second and subsequent aspects of the inventionare as for the first aspect mutatis mutandis.

BRIEF DESCRIPTION OF THE DRAWINGS

The invention will now be further described by reference to thefollowing examples and drawings which are provided for the purpose ofexplanation only and should not be construed as being limiting on thepresent invention. In the examples, reference is made to a number ofdrawings in which:

FIG. 1 shows the nucleotide sequence of the P. coccineus 2-oxidase cDNAclone pc-2boh.dna (PcGA2ox1) (SEQ ID NO:1) with the coding region atresidues 68-1063nt (332 amino acids).

FIG. 2 shows the deduced amino acid sequence for the P. coccineusnucleotide sequence (PcGA2ox1) (SEQ ID NO:2) shown in FIG. 1.

FIGS. 3a-3 b show the DNA probe sequences for A. thaliana probe T3 (SEQID NO:3) (FIG. 3a) and probe T24 (SEQ ID NO:4) (FIG. 3b).

FIG. 4 shows the two major pathways of gibberellin (GA) biosynthesis.

FIG. 5 shows the partial nucleotide sequence for A. thaliana cloneat-2bt3 (AtGA2ox1) (SEQ ID NO:5) with the coding region at residues41-1027nt (329 amino acids).

FIG. 6 shows the deduced amino acid sequence for A. thaliana cloneat-2bt3 (AtGA2ox1) (SEQ ID NO:6).

FIG. 7 shows the partial nucleotide sequence for A. thaliana cloneat-2bt24 (AtGA2ox2) (SEQ ID NO:7), with the coding region at residues109-1131nt (341 amino acids).

FIG. 8 shows the deduced amino acid sequence for A. thaliana cloneat-2bt24 (AtGA2ox2) (SEQ ID NO:8).

FIG. 9 shows the nucleotide sequence for A. thaliana genomic cloneT31E10.11 (AtGA2ox3) (SEQ ID NO:9).

FIG. 10 shows the deduced amino acid sequence for genomic cloneT31E10.11 (AtGA2ox3) (SEQ ID NO:10).

FIG. 11 shows a photograph of transformed Arabidopsis plants (Columbiaecotype) expressing P. coccineus GA 2-oxidase cDNA under control of CaMV35S promoter, including a transformed plant showing no phenotype(extreme right).

EXAMPLES Example 1 Isolation of cDNA Clone Encoding GA 2β-hydroxylaseFrom Phaseolus coccineus

A cDNA clone encoding a GA 2β-hydroxylase was isolated from Phaseoluscoccineus embryos by screening a cDNA library for expression offunctional enzyme as follows. RNA was extracted from the cotyledons ofmature Phaseolus coccineus seeds, according to Dekker et al. (1989).Poly (A)+ mRNA was purified by chromatography on oligo(dt) cellulose.cDNA was synthesized from 5 μg of poly(A)+ mRNA using a directional cDNAsynthesis kit (λ-ZAP II cDNA synthesis kit, Stratagene). The cDNA wasligated into λ-ZAP II arms, packaged using Gigapack Gold III(Stratagene) and 1×10⁶ recombinant clones amplified according to themanufacturer's instructions.

A phagemid stock was prepared from the Phaseolus coccineus cDNA library(1×10⁹ pfu) according to the manufacturer's in vivo excision protocol(Stratagene). For the primary screen, E. coli SOLR were infected withthe phagemid stock according to the manufacturer's instructions(Stratagene), resulting in approximately 11000 colony forming units(cfu). These were subdivided into 48 wells (6×8 array) of a micro-titreplate (well volume=3.5 ml) and amplified by overnight growth at 37° C.with shaking, in 0.5 ml of 2×YT broth supplemented with 50 μg/mlkanamycin and 100 μg/ml carbenicillin. Aliquots (20 μl) from the sixwells in each row, and from the eight wells in each column were combinedto make 14 pools and each added to 10 ml 2YT broth, supplemented with 50μg/ml kanamycin and 100 μg/ml carbenicillin, and grown at 37° C. withshaking until an OD 600 nm of 0.2-0.5. The cultures were thentransferred to a 30° C. shaking incubator and recombinant fusion proteinproduction induced by the addition of IPTG to 1 mM. Cultures wereinduced for 16 hours. The bacteria were pelleted by centrifugation (3000g×10 min) and resuspended in 750 μl of lysis buffer (100 mM Tris HCl pH7.5, 5 mM DTT). Bacteria were lysed by sonication (3×10 s) and the celldebris pelleted by centrifugation for 10 min in a microfuge. Thesupernatants were assayed for GA 2β-hydroxylase activity as describedbelow. Cell lysates from pooled bacteria of row 6 (R6) and column 1 (C1)were capable of catalysing the release of ³H₂O from [1,2-³H₂]GA₄ and[2,3-³H₂]GA₉. For the secondary screen bacteria from well R6C1 wereplated out on 2YT agar plates, supplemented with 100 μg./mlcarbenicillin and 50 μg/ml kanamycin, and grown for 16 hours at 37° C.One hundred single colonies were picked at random and transferred to 5ml 2×YT broth containing 100 mg/ml carbenicillin and 50 mg/ml kanamycinand grown, with shaking, for 16 hours at 37° C. The cultures werearranged in a 10×10 grid and pools from each row and column induced andtested for GA 2β-hydroxylase activity as described above. Rows 2 and 9and columns 7 and 10 were capable of catalysing the release of ³H₂O from[1,2-³H₂]GA₄ and [2,3-³H₂]GA₉. Cultures 27 and 90 were shown to beresponsible for this activity. The putative GA 2β-hydroxylase clone wasdesignated as 2B27.

Plasmid DNA, isolated from clone 2B27 using the Promega SV miniprep kit,was sequenced using Amersham's Taq cycle sequencing kit with the M13universal (−20) and reverse sequencing primers. The chain terminationproducts produced from the sequencing reactions were analysed using anApplied Biosystems 373A automated sequencer. Sequence analysis wasperformed using the program Sequencer 3.0 from Gene Codes Corporation.Further nucleotide and protein sequence analyses were performed usingthe University of Wisconsin Genetics Computer Group suite of programs.

Example 2 Assays of GA 2-oxidase Activity

GA 2β-hydroxylase activity was determined by measuring the release of³H₂O from a 2β tritiated GA substrate, as described by Smith andMacMillan (Smith, V. A., and MacMillan, J. in J. Plant Growth Regulation2 251-264 (1984)). The bacterial lysate (90 μl) was incubated with[1,2-³H₂]GA₄ or [2,3-³H₂]GA₉ (ca. 50000 dpm), in the presence of 4 mM2-oxoglutarate, 0.5 mM Fe(II)SO₄, 4 mM ascorbate, 4 mM DTT, 1 mg/mlcatalase, 2 mg/ml BSA, in a final reaction volume of 100 μl. The mixturewas incubated at 30° C. for 60 min. The tritiated GAs were removed bythe addition of 1 ml of activated charcoal (5% w/v) and subsequentcentrifugation for 5 min in a microfuge. Aliquots (0.5 ml) of thesupernatant were mixed with 2 ml scintillation fluid and theradioactivity determined by scintillation counting.

In order to confirm the function of the cDNA expression products,bacterial lysate was incubated with [17-¹⁴C]GAs in the presence ofcofactors, as described above. After the incubation, acetic acid (10 μl)and water at pH 3 (140 μl) were added, and the mixture was centrifugedat 3,000 rpm for 10 min. The supernatant was analysed by HPLC withon-line radiomonitoring and products identified by GC-MS, as describedpreviously (MacMillan et al Plant Physiol. 113 1369-1377 (1997)).

Example 3 Cloning of cDNAs Encoding GA 2-oxidase From Arabidopsisthaliana

The predicted protein sequence of clone 2B27 was used to search theGenomic Survey Sequences database at the National Centre for BiologicalInformation (ncbi.nlm.nih.gov) using the TblastN program. TwoArabidopsis genomic sequences, T3M9-Sp6 and T24E24TF, demonstrated highamino acid sequence identity with the 2B27 sequence. Oligonucleotideprimers were designed based on the T3M9-Sp6 genomic sequences:

5′-TAATCACTATCCACCATGTC-3′ (sense) (SEQ ID NO:11),

5′-TGGAGAGAGTCACCCACGTT (antisense) (SEQ ID NO:12), and

T24E24TF sequences:

5′-GGTTATGACTAACGGGAGGT-3′ (sense) (SEQ ID NO:13),

5′-CTTGTAAGCAGAAGATTTGT-3′ (antisense) (SEQ ID NO:14),

and used in PCR reactions with Arabidopsis genomic DNA as a template.The PCR reactions consisted of 200 ng of genomic DNA, 1×PCR buffer, 1.5mM MgCl₂, 200 μM deoxynucleoside triphosphates, 1 μM of each primer and2 units of Taq DNA polymerase (Promega). The reactions were heated to94° C. for 3 min then 35 cycles of amplification were performed (94° C.for 30 seconds, 55° C. for 30 seconds and 72° C. for 30 seconds)followed by a final 10 min incubation at 72° C. Resulting PCR productswere cloned directly into the pCR2.1 vector using the TA cloning kit(Invitrogen) and sequenced as described above. The clones weredesignated as AtT3 and AtT24. Siliques, flowers, upper stems (the top 2cm of stem), lower sterns, leaves (cauline and rosette) and roots of theColumbia ecotype were collected and frozen in liquid N₂. Poly(A)⁺ mRNAwas extracted as described above. Northern blots were prepared by theelectrophoresis of 5 μg samples of the poly(A)⁺ mRNA through agarosegels containing formaldehyde and subsequent transfer to nitrocellulose(Sambrook et al Molecular Cloning: A Laboratory Manual. Cold SpringHarbor Laboratory Press, Plainview, N.Y. (1989)). Random-primed³²P-labelled probes were generated for AtT3 and AtT24 using Ready to golabelling beads (Pharmacia). FIG. 3 shows the DNA probe sequences for A.thaliana probe T3 (FIG. 3a) and probe T24 (FIG. 3b). Hybridisations werecarried out in the presence of 50% formamide at 42° C. for 16 h(hybridisation buffer:5×SSPE, 2×Denhardts, 0.5% (w/v) SDS, 100 μg/mldenatured sonicated salmon sperm DNA, 10% Dextran sulphate). Blots werewashed twice for 10 min in 1×SSC/0.5% SDS at 20° C. A further 2×10 minwashes were performed in 0.1×SSC/0.5% SDS at 60° C. Blots were exposedto Kodak MS film at −80° C. with MS intensifying screens: highestexpression of both genes was detected in the inflorescence. A cDNAlibrary was constructed using 5 μg of inflorescence poly(A)⁺ mRNA asdescribed above. A total of 5×10⁵ recombinant phage in E. coli XL1-BlueMRF′ were plated on five 24 cm×24 cm square plates. Plaques were grownuntil confluency (8-10 h), then duplicate lifts were taken on 20×22 cmsupported nitrocellulose filters (Nitropure, MSI) and processed asdescribed by Sambrook et al (Molecular Cloning: A Laboratory Manual.Cold Spring Harbor Laboratory Press, Plainview, N.Y. (1989)).Hydridization of ³²P-labelled AtT3 and AtT24 probes was performed asdescribed above. Positive plaques were identified by autoradiography andcored from the plates into 750 μl of SM buffer (50 mM Tris HCl pH7.5,100 mM NaCl, 10 mM MgSO₄, 0.5% gelatine) and rescreened untilplaque-pure clones were isolated. Plasmid rescue was performed usingStratagene's Rapid Excision kit. The cDNA clones were sequenced andrecombinant protein expressed in E. coli and tested for GA 2-oxidaseactivity as described above. The partial nucleotide and deduced aminoacid sequences for the clones are shown in FIGS. 5, 6, 7 and 8.

A third Arabidopsis genomic sequence T31E10.11 (AtGA2ox3), with a highamino acid identity with the P. coccineus GA 2-oxidase (PcGA2ox1) wasalso detected in the GenBank database. Its derived amino acid sequencehas 53%, 49% and 67% identity (67%, 67% and 84% similarity) with the P.coccineus GA 2-oxidase (PcGA2ox1), T3 (AtGA2ox1), and T24 (AtGA2ox2),respectively. The nucleotide sequence of T31 is shown in FIG. 9 and thededuced amino acid sequence is shown in FIG. 10.

Example 4 Transformation of Arabidopsis With Sense and Antisense GA2-oxidase cDNA Constructs

The predicted coding region of 2B27 was amplified by PCR usingoligonucleotide primers:

5′-TGAGCTCAACCATGGTTGTTCTGTCTCAGC-3′ (sense) (SEQ ID NO:15), and

5′-TGAGCTCTTAATCAGCAGCAGATTTCTGG-3′ (antisense) (SEQ ID NO:16),

each of which had a SacI restriction site incorporated at its 5′ end.The PCR product was sub-cloned into pCR2.1 to facilitate DNA sequencingas described previously.

The 2B27 coding region was digested with SacI and sub-cloned into SacIsite of the binary vector pLARS120, a modified version of pGPTV-Kan(Becker et al. Plant Mol Biol 20 1195-1197 (1992)) in which theβ-glucuronidase reporter gene is replaced by the cauliflower mosaicvirus 35S promoter from pBI220 (Jefferson, R. A., Plant Mol Biol Rep 5387405 (1987)). The DNA was inserted in the sense orientation under thecontrol of the 35S promoter. The plasmid was introduced intoAgrobacterium tumefaciens by electroporation and then transferred intoArabidopsis cv. Columbia via a vacuum infiltration method (Bechtold etal. Compt. Rend. Acad. Sci. Serie iii-Sciences de la Vie-Life Science316 1194-1199 (1993)). Similarly, SacI fragments of AtT3 and AtT24 weresub-cloned into pLARS120, except in these two cases the DNA was insertedin the antisense orientation under the control of the 35S promoter.Arabidopsis was transformed with these two antisense constructs asdescribed above.

Example 5(a) Altered Expression of GA 2-oxidase in Transgenic Plants

The P. coccineus 2-oxidase cDNA in sense orientation (PcGA2ox1) and theA. thaliana 2-oxidase cDNA in antisense orientations were insertedbetween the CaMV 35S promoter and nos terminator in the vector pLARS120.The vector pLARS120 is a binary vector for Agrobacterium-mediated planttransformation: the T-DNA contains, in addition to the CaMV 35S promoterand nos terminator, an nptII selectable marker under the nos promoter.The vector is derived from pGPTV-Kan (Becker et al Plant Mol. Biol. 201195-1197 (1992)), the uidA reporter gene in pGPTV-Kan being replaced bythe 35S promoter. The binary expression constructs were introduced intoAgrobacterium tumefaciens strain GV3101 carrying the pAD1289 plasmidconferring overexpression of VirG by electroporation. These wereintroduced into Arabidopsis by the vacuum infiltration method (Bechtoldet al Compt. Rend. Acad. Sci. Serie iii-Sciences de la Vie-Life Science316 1194-1199 (1993)). To identify transgenic plants, seeds frominfiltrated plants were grown on MS plates supplemented with kanamycin(50 μg/ml) for approximately 14 days and resistant plants weretransferred to compost. T-DNA containing the P. coccineus 2-oxidaseconstruct was also introduced into Nicotiana sylvestris by the infectionof leaf discs with transformed Agrobacterium tumefaciens.

The transformation of Arabidopsis plants with P. coccineus GA 2-oxidasecDNA under the control of the CAMV 35S promoter yielded the followingresults. Over half of all transformants examined showed some degree ofdwarfing, many were severely dwarfed and some failed to bolt. FIG. 11shows a photograph of a selection of dwarf transformed plants comparedwith a transformed plant showing no phenotype (Columbia ecotype). Thetransformants responded to treatment with GA₃ with increased stemelongation and normal flower development so that it was possible toobtain seeds. Overexpression of the P. coccineus GA 2-oxidase cDNA inNicotiana sylvestris resulted in plants with reduced stem height. Onetransformant did not bolt or produce flowers, whereas non-transformedplants of the same age had already flowered.

Example 5(b) Altered Expression of GA 2-oxidase in Transgenic Plants

Five Arabidopsis lines that are homozygous for the 35S-PcGA2ox1transgene were obtained. One line bolted at the same time as wild-type(Columbia) plants but had reduced stem height, whereas the other fourlines remained as rosettes and did not bolt when grown in long (16 hour)or short (10 hour) photoperiods. One severely dwarfed line has failed toproduce homozygous plants, even when seeds were germinated in thepresence of GA₃, indicating that seed development was impaired in thisline when two copies of the gene were present. The severely dwarfedlines possessed small, dark leaves that remained close to coil level.They produced flower buds when grown in long photoperiods although theflowers did not develop normally and were infertile. No flower buds wereobtained when plants were grown in short photoperiods. Treatment of thetransgenic lines with 10 μM GA₃ enabled them to bolt and produce normalflowers that set viable seed.

Metabolism of C₁₉-Gas was compared in a severely dwarfed 35A-PcGA2ox1line with that in wild-type plants. On the basis of HPLC, the dwarf lineconverted GA₁, GA₄, GA₉ and GA₂₀ to 2-oxidised products to a muchgreater extent than did the wild-type. The dwarf line did not metaboliseGA₃, confirming results with recombinant enzyme indicating that GA₃ isnot a substrate for the GA 2-oxidases. Therefore, this GA can be used,when required, to reverse the GA-deficiency resulting fromoverexpressing GA 2-oxidase genes.

Northern blot analysis of the 35S-PcGA2ox1 lines confirmed high levelsof expression of the transgene. Transcript abundance for GA 20-oxidase(AtGA2ox1) and 3β-hydroxylase (AtGA3ox1) genes was elevated in rosettesof the 35S-PcGA2ox1 lines compared with wild-type plants, whereas nativeGA 2-oxidase (AtGA2ox2) transcript level was reduced, as a consequenceof the control mechanisms for GA homeostasis.

Example 6 Expression Patterns of Arabidopsis GA 2-oxidase Genes T3 andT24 (AtGA2ox1 and AtGA2ox2 Respectively)

The expression patterns of Arabidopsis GA 2-oxidase genes T3 and T24were examined by probing Northern blots of RNA extracted from differenttissues with the full length cDNAs. The genes showed similar patterns ofexpression, with transcript for both genes present in leaves, lowerstems, upper stems, flowers and siliques. The highest levels ofexpression were in flowers, siliques and upper stems in decreasing orderof transcript abundance. T24, but not T3, was also expressed in roots.Transcript abundance for both T3 and T24 in immature flower buds andpedicels of the GA-deficient Arabidopsis mutant, gal-2, is increasedafter treatment with GA₃, indicating that expression of these 2-oxidasegenes is upregulated by GA. This contrasts with expression of the GA20-oxidase and 3β-hydroxylase genes which are downregulated by GA.Transcript abundance for T31 was much lower in all tissues than for T3or T24. T31 transcript was detected by RT-PCR in flowers, upper stemsand leaves but not in roots or siliques.

Example 7 Function of the Recombinant GA 2-oxidases From Phaseoluscoccineus and Arabidopsis thaliana

The catalytic properties of the recombinant proteins obtained byexpressing the cDNAs from P. coccineus (PcGA2ox1) and A. thaliana(AtGA2ox1, AtGA2ox2 and AtGa2ox3) in E. coli were examined by incubatingin the presence of dioxygenase cofactors with a range of ¹⁴C-labelled GAsubstrates, consisting of the C₁₉-GAs GA₁, GA₄, GA₉, and GA₂₀, and theC₂₀-GAs, GA₁₂ and GA₁₅. This last compound was incubated in both itsclosed and open lactone forms. No conversion of GA₁₂ was obtained withany of the enzymes, whereas GA₁₅ was converted to a single product byPcGA2ox1 and AtGA2ox2. The open lactone form of GA₁₅ (20-hydroxyGA₁₂)was converted to the same product by AtGAox2, but less efficiently thanwas the lactone form, whereas there was no conversion of GA₁₅ openlactone by PcGA2ox1. The mass spectrum of the product from GA₁₅ isconsistent with it being 2β-hydroxyGA₁₅, although, because the authenticcompound is not available for comparison, the identity of this productis tentative.

Comparison of the substrate specificities of the recombinant enzymes forthe C₁₉-GAs (Table 1) indicated that GA₉ was the preferred substrate forPcGA2ox1, AtGA2ox1 and AtGA2ox2. The recombinant enzymes differedsomewhat in their substrate specificities, with GA₄ being converted aseffectively as GA₉ by PcGA2ox1 and AtGA2ox3, but a relatively poorsubstrate for AtGA2ox1 and AtGA2ox2. Although GA₂₀ was 2β-hydroxylatedmore efficiently than GA₄ by AtGA2ox1 and AtGA2ox2, no GA₂₉ catabolitewas detected after incubations with GA₂₀, whereas low yields of GA₃₄catabolite were obtained when GA₄ was incubated with PcGA2ox1, AtGA2ox2and AtGA2ox3. The activities of recombinant PcGA2ox1, AtGA2ox2 andAtGA2ox3 for 2β-hydroxylation of GA₉ varied little between pH 6.5 and 8,and that of AtGA2ox1 peaked at pH 7 with no detectable activity atpH≦5.9 and≧8.1.

The results indicate that the non-3β-hydroxy C₁₉-GAs, which areimmediate precursors of the biologically active compounds, are bettersubstrates for the GA 2-oxidases than are the active Gas themselves.Therefore, overexpression of GA 2-oxidase genes would result in verylittle active GA being produced.

TABLE 1 Specificity of recombinant GA 2-oxidase for C₁₉-GA substratesRecombinant ¹⁴C-labelled GA 2β-Hydroxy GA GA-catabolite Enzymessubstrate product Product PcGA2ox1 GA₁  100  — GA₄  83 17 GA₉  87 13GA₂₀ 86 — AtGA2ox1 GA₁  41 — GA₄  25 — GA₉  91 — GA₂₀ 50 — AtGA2ox2 GA₁ 100  — GA₄  77 23 GA₉  — 100  GA₂₀ 100  — AtGA2ox3 GA₁  100  Trace GA₄ 86 14 GA₉  100  — GA₂₀ 25 —

Values are % yield by HPLC-radiomonitoring of products after incubationof cell lysates from E. coli expressing the cDNA with ¹⁴C-labelled GAsubstrate and cofactors for 2.5 h. Products and substrate were separatedby HPLC and products identified by GC-MS. Where combined yield ofproducts <100%, the remainder is unconverted substrate.

Gibberellin (GA) Biosynthesis

FIG. 4 shows the two major pathways of gibberellin (GA) biosynthesis,from GA₁₂ to GA₄ and from GA₅₃ to GA₁. GA₁ and GA₄ are the biologicallyactive GAs. The conversion of GA₁₂ to GA₉ and of GA₅₃ to GA₂₀ arecatalysed by GA 20-oxidase. The conversion of GA₉ to GA₄ and of GA₂₀ toGA₁ are catalysed by GA 3β-hydroxylase. GA₉, GA₄, GA₂₀ and GA₁ are allsubstrates for the 2β-hydroxylase activity of GA 2-oxidase, beingconverted to GA₅₁, GA₃₄, GA₂₉ and GA₈ respectively. These2β-hydroxylated GAs can be further oxidised to the correspondingcatabolites. The present invention shows that the enzyme from P.coccineus and the two enzymes from Arabidopsis thaliana catalyse the2β-hydroxylation of each substrate. In addition, the present inventionshows that the P. coccineus enzyme and one of the A. thaliana enzymesforms GA₅₁-catabolite and GA₃₄-catabolite when incubated with GA₉ andGA₄ respectively.

16 1 1318 DNA Phaseolus coccineus 1 gtttctcttc cttaccctgt tctgcttctctttttcatag taacaatcga caacaacaac 60 aacaaccatg gttgttctgt ctcagccagcattgaaccag tttttccttc tgaaaccatt 120 caagtccacg cccttgttca cggggattcctgtggtcgac ctcacgcacc ccgatgccaa 180 gaatctcata gtgaacgcct gtagggacttcggcttcttc aagcttgtga accatggtgt 240 tccattggag ttaatggcca atttagaaaacgaggccctc aggttcttta aaaaatctca 300 gtccgagaaa gacagagctg gtccccccgaccctttcggc tatggtagca agaggattgg 360 cccaaacggt gatgtcggtt gggtcgaatacctcctcctc aacaccaacc ctgatgttat 420 ctcacccaaa tcactttgca ttttccgagaaaatcctcat catttcaggg cggtggtgga 480 gaactacatt acagcagtga agaacatgtgctatgcggtg ttggaattga tggcggaggg 540 gttggggata aggcagagga atacgttaagcaggttgctg aaggatgaga aaagtgattc 600 gtgcttcagg ttgaaccact acccgccttgccctgaggtg caagcactga accggaattt 660 ggttgggttt ggggagcaca cagacccacagataatttct gtcttaagat ctaacagcac 720 atctggcttg caaatctgtc tcacagatggcacttgggtt tcagtcccac ctgatcagac 780 ttcctttttc atcaatgttg gtgacgctctacaggtaatg actaatggga ggtttaaaag 840 tgtaaagcat agggttttgg ctgacacaacgaagtcaagg ttatcaatga tctactttgg 900 aggaccagcg ttgagtgaaa atatagcacctttaccttca gtgatgttaa aaggagagga 960 gtgtttgtac aaagagttca catggtgtgaatacaagaag gctgcgtaca cttcaaggct 1020 agctgataat aggcttgccc ctttccagaaatctgctgct gattaaccaa acacaccctt 1080 caaattccac tcattttacg cacgtgttattaccccaatt ttctttcctt tttcttttcc 1140 tgtgtctgtc taggtttcaa acagttgactctacttgaca tatatagaaa atgaataggt 1200 taagatgttt atcattttct ttttcttgtttcatctaagt gtaacagttg gtctcaactt 1260 ccctttcctc aattgtcaat ggaacgcaactctagttaca aaaaaaaaaa aaaaaaaa 1318 2 331 PRT Phaseolus coccineus 2 MetVal Val Leu Ser Gln Pro Ala Leu Asn Gln Phe Phe Leu Leu Lys 1 5 10 15Pro Phe Lys Ser Thr Pro Leu Phe Thr Gly Ile Pro Val Val Asp Leu 20 25 30Thr His Pro Asp Ala Lys Asn Leu Ile Val Asn Ala Cys Arg Asp Phe 35 40 45Gly Phe Phe Lys Leu Val Asn His Gly Val Pro Leu Glu Leu Met Ala 50 55 60Asn Leu Glu Asn Glu Ala Leu Arg Phe Phe Lys Lys Ser Gln Ser Glu 65 70 7580 Lys Asp Arg Ala Gly Pro Pro Asp Pro Phe Gly Tyr Gly Ser Lys Arg 85 9095 Ile Gly Pro Asn Gly Asp Val Gly Trp Val Glu Tyr Leu Leu Leu Asn 100105 110 Thr Asn Pro Asp Val Ile Ser Pro Lys Ser Leu Cys Ile Phe Arg Glu115 120 125 Asn Pro His His Phe Arg Ala Val Val Glu Asn Tyr Ile Thr AlaVal 130 135 140 Lys Asn Met Cys Tyr Ala Val Leu Glu Leu Met Ala Glu GlyLeu Gly 145 150 155 160 Ile Arg Gln Arg Asn Thr Leu Ser Arg Leu Leu LysAsp Glu Lys Ser 165 170 175 Asp Ser Cys Phe Arg Leu Asn His Tyr Pro ProCys Pro Glu Val Gln 180 185 190 Ala Leu Asn Arg Asn Leu Val Gly Phe GlyGlu His Thr Asp Pro Gln 195 200 205 Ile Ile Ser Val Leu Arg Ser Asn SerThr Ser Gly Leu Gln Ile Cys 210 215 220 Leu Thr Asp Gly Thr Trp Val SerVal Pro Pro Asp Gln Thr Ser Phe 225 230 235 240 Phe Ile Asn Val Gly AspAla Leu Gln Val Met Thr Asn Gly Arg Phe 245 250 255 Lys Ser Val Lys HisArg Val Leu Ala Asp Thr Thr Lys Ser Arg Leu 260 265 270 Ser Met Ile TyrPhe Gly Gly Pro Ala Leu Ser Glu Asn Ile Ala Pro 275 280 285 Leu Pro SerVal Met Leu Lys Gly Glu Glu Cys Leu Tyr Lys Glu Phe 290 295 300 Thr TrpCys Glu Tyr Lys Lys Ala Ala Tyr Thr Ser Arg Leu Ala Asp 305 310 315 320Asn Arg Leu Ala Pro Phe Gln Lys Ser Ala Ala 325 330 3 210 DNA ArtificialSequence Description of Artificial Sequence Probe 3 taatcactatccaccatgtc ctcttagcaa taagaaaacc aatggtggta agaatgtgat 60 tggttttggtgaacacacag atcctcaaat catctctgtc ttaagatcta acaacacttc 120 tggtctccaaattaatctaa atgatggctc atggatctct gtccctcccg atcacacttc 180 cttcttcttcaacgtgggtg actctctcca 210 4 199 DNA Artificial Sequence Description ofArtificial Sequence Probe 4 ggttatgact aacgggaggt tcaagagtgt taaacacagggtcttagccg atacaaggag 60 atcgaggatt tcaatgatat atttcggcgg accgccattgagccagaaga tcgcaccatt 120 gccatgcctt gtccctgagc aagatgattg gctttacaaagaattcactt ggtctcaata 180 caaatcttct gcttacaag 199 5 1318 DNAArabidopsis thaliana misc_feature (1243, 1265) unidentified residue 5tcaaaatcaa aaaaattcta tcaaacaagg aaatatatca atggcggtat tgtctaaacc 60ggtagcaata ccaaaatccg ggttctctct aatcccggtt atagatatgt ctgacccaga 120atccaaacat gccctcgtga aagcatgcga agacttcggc ttcttcaagg tgatcaacca 180tggcgtttcc gcagagctag tctctgtttt agaacacgag accgtcgatt tcttctcgtt 240gcccaagtca gagaaaaccc aagtcgcagg ttatcccttc ggatacggga acagtaagat 300tggtcggaat ggtgacgtgg gttgggttga gtacttgttg atgaacgcta atcatgattc 360cggttcgggt ccactatttc caagtcttct caaaagcccg ggaactttca gaaacgcatt 420ggaagagtac acaacatcag tgagaaaaat gacattcgat gttttggaga agatcacaga 480tgggctaggg atcaaaccga ggaacacact tagcaagctt gtgtctgacc aaaacacgga 540ctcgatattg agacttaatc actatccacc atgtcctctt agcaataaga aaaccaatgg 600tggtaagaat gtgattggtt ttggtgaaca cacagatcct caaatcatct ctgtcttaag 660atctaacaac acttctggtc tccaaattaa tctaaatgat ggctcatgga tctctgtccc 720tcccgatcac acttccttct tcttcaacgt tggtgactct ctccaggtga tgacaaatgg 780gaggttcaag agcgtgaggc atagggtttt agctaactgt aaaaaatcta gggtttctat 840gatttacttc gctggacctt cattgactca gagaatcgct ccgttgacat gtttgataga 900caatgaggac gagaggttgt acgaggagtt tacttggtct gaatacaaaa actctaccta 960caactctaga ttgtctgata ataggcttca acaattcgaa aggaagacta taaaaaatct 1020cctaaattga tgtgatatat ctatttaatc tataagtgtg tgctacatac agacaatgca 1080tctgtatatt ttgaagtata atgttatttg ttaatccaat aactgtaaaa acatgcaaga 1140gtgtgtttgt ttgtttcgta atatcaacat cgctcccatc ttttatggat aaaaaaaaaa 1200aaaaaaaaaa cactgttttg atgtaagcta cattttactt tangtgtaca tcttattgtg 1260ttaantaaat tatttcaaaa taaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaa 1318 6329 PRT Arabidopsis thaliana 6 Met Ala Val Leu Ser Lys Pro Val Ala IlePro Lys Ser Gly Phe Ser 1 5 10 15 Leu Ile Pro Val Ile Asp Met Ser AspPro Glu Ser Lys His Ala Leu 20 25 30 Val Lys Ala Cys Glu Asp Phe Gly PhePhe Lys Val Ile Asn His Gly 35 40 45 Val Ser Ala Glu Leu Val Ser Val LeuGlu His Glu Thr Val Asp Phe 50 55 60 Phe Ser Leu Pro Lys Ser Glu Lys ThrGln Val Ala Gly Tyr Pro Phe 65 70 75 80 Gly Tyr Gly Asn Ser Lys Ile GlyArg Asn Gly Asp Val Gly Trp Val 85 90 95 Glu Tyr Leu Leu Met Asn Ala AsnHis Asp Ser Gly Ser Gly Pro Leu 100 105 110 Phe Pro Ser Leu Leu Lys SerPro Gly Thr Phe Arg Asn Ala Leu Glu 115 120 125 Glu Tyr Thr Thr Ser ValArg Lys Met Thr Phe Asp Val Leu Glu Lys 130 135 140 Ile Thr Asp Gly LeuGly Ile Lys Pro Arg Asn Thr Leu Ser Lys Leu 145 150 155 160 Val Ser AspGln Asn Thr Asp Ser Ile Leu Arg Leu Asn His Tyr Pro 165 170 175 Pro CysPro Leu Ser Asn Lys Lys Thr Asn Gly Gly Lys Asn Val Ile 180 185 190 GlyPhe Gly Glu His Thr Asp Pro Gln Ile Ile Ser Val Leu Arg Ser 195 200 205Asn Asn Thr Ser Gly Leu Gln Ile Asn Leu Asn Asp Gly Ser Trp Ile 210 215220 Ser Val Pro Pro Asp His Thr Ser Phe Phe Phe Asn Val Gly Asp Ser 225230 235 240 Leu Gln Val Met Thr Asn Gly Arg Phe Lys Ser Val Arg His ArgVal 245 250 255 Leu Ala Asn Cys Lys Lys Ser Arg Val Ser Met Ile Tyr PheAla Gly 260 265 270 Pro Ser Leu Thr Gln Arg Ile Ala Pro Leu Thr Cys LeuIle Asp Asn 275 280 285 Glu Asp Glu Arg Leu Tyr Glu Glu Phe Thr Trp SerGlu Tyr Lys Asn 290 295 300 Ser Thr Tyr Asn Ser Arg Leu Ser Asp Asn ArgLeu Gln Gln Phe Glu 305 310 315 320 Arg Lys Thr Ile Lys Asn Leu Leu Asn325 7 1237 DNA Arabidopsis thaliana 7 gaattcggca cgagtttcct tcttcttcctcaacctttgc ttcaatcttc aacaactttc 60 tttttataaa gattttgcaa gttaagtgtaaacctacaaa aaccaaacat ggtggttttg 120 ccacagccag tcactttaga taaccacatctccctaatcc ccacatacaa accggttccg 180 gttctcactt cccattcaat ccccgtcgtcaacctagccg atccggaagc gaaaacccga 240 atcgtaaaag cctgcgagga gttcgggttcttcaaggtcg taaaccacgg agtccgaccc 300 gaactcatga ctcggttaga gcaggaggctattggcttct tcggcttgcc tcagtctctt 360 aaaaaccggg ccggtccacc tgaaccgtacggttatggta ataaacggat tggaccaaac 420 ggtgacgttg gttggattga gtatctcctcctcaatgcta atcctcagct ctcctctcct 480 aaaacctccg ccgttttccg tcaaacccctcaaattttcc gtgagtcggt ggaggagtac 540 atgaaggaga ttaaggaagt gtcgtacaaggtgttggaga tggttgccga agaactaggg 600 atagagccaa gggacactct gagtaaaatgctgagagatg agaagagtga ctcgtgcctg 660 agactaaacc attatccggc ggcggaggaagaggcggaga agatggtgaa ggtggggttt 720 ggggaacaca cagacccaca gataatctcagtgctaagat ctaataacac ggcgggtctt 780 caaatctgtg tgaaagatgg aagttgggtcgctgtccctc ctgatcactc ttctttcttc 840 attaatgttg gagatgctct tcaggttatgactaacggga ggttcaagag tgttaaacac 900 agggtcttag ccgatacaag gagatcgaggatttcaatga tatatttcgg cggaccgcca 960 ttgagccaga agatcgcacc attgccatgccttgtccctg agcaagatga ttggctttac 1020 aaagaattca cttggtctca atacaaatcttctgcttaca agtctaagct tggtgattat 1080 agacttggtc tctttgagaa acaacctcttctcaatcata aaacccttgt atgagagtag 1140 tcatgatgat ctttatcatc ctttgtacgatagaaagtca taatcacaaa aagaaggaaa 1200 tggatagtgt tttggattaa aaaaaaaaaaaaaaaaa 1237 8 341 PRT Arabidopsis thaliana 8 Met Val Val Leu Pro GlnPro Val Thr Leu Asp Asn His Ile Ser Leu 1 5 10 15 Ile Pro Thr Tyr LysPro Val Pro Val Leu Thr Ser His Ser Ile Pro 20 25 30 Val Val Asn Leu AlaAsp Pro Glu Ala Lys Thr Arg Ile Val Lys Ala 35 40 45 Cys Glu Glu Phe GlyPhe Phe Lys Val Val Asn His Gly Val Arg Pro 50 55 60 Glu Leu Met Thr ArgLeu Glu Gln Glu Ala Ile Gly Phe Phe Gly Leu 65 70 75 80 Pro Gln Ser LeuLys Asn Arg Ala Gly Pro Pro Glu Pro Tyr Gly Tyr 85 90 95 Gly Asn Lys ArgIle Gly Pro Asn Gly Asp Val Gly Trp Ile Glu Tyr 100 105 110 Leu Leu LeuAsn Ala Asn Pro Gln Leu Ser Ser Pro Lys Thr Ser Ala 115 120 125 Val PheArg Gln Thr Pro Gln Ile Phe Arg Glu Ser Val Glu Glu Tyr 130 135 140 MetLys Glu Ile Lys Glu Val Ser Tyr Lys Val Leu Glu Met Val Ala 145 150 155160 Glu Glu Leu Gly Ile Glu Pro Arg Asp Thr Leu Ser Lys Met Leu Arg 165170 175 Asp Glu Lys Ser Asp Ser Cys Leu Arg Leu Asn His Tyr Pro Ala Ala180 185 190 Glu Glu Glu Ala Glu Lys Met Val Lys Val Gly Phe Gly Glu HisThr 195 200 205 Asp Pro Gln Ile Ile Ser Val Leu Arg Ser Asn Asn Thr AlaGly Leu 210 215 220 Gln Ile Cys Val Lys Asp Gly Ser Trp Val Ala Val ProPro Asp His 225 230 235 240 Ser Ser Phe Phe Ile Asn Val Gly Asp Ala LeuGln Val Met Thr Asn 245 250 255 Gly Arg Phe Lys Ser Val Lys His Arg ValLeu Ala Asp Thr Arg Arg 260 265 270 Ser Arg Ile Ser Met Ile Tyr Phe GlyGly Pro Pro Leu Ser Gln Lys 275 280 285 Ile Ala Pro Leu Pro Cys Leu ValPro Glu Gln Asp Asp Trp Leu Tyr 290 295 300 Lys Glu Phe Thr Trp Ser GlnTyr Lys Ser Ser Ala Tyr Lys Ser Lys 305 310 315 320 Leu Gly Asp Tyr ArgLeu Gly Leu Phe Glu Lys Gln Pro Leu Leu Asn 325 330 335 His Lys Thr LeuVal 340 9 1008 DNA Arabidopsis thaliana 9 atggtaattg tgttacagccagccagtttt gatagcaacc tctatgttaa tccaaaatgc 60 aaaccgcgtc cggttttaatccctgttata gacttaaccg actcagatgc caaaacccaa 120 atcgtcaagg catgtgaagagtttgggttc ttcaaagtca tcaaccatgg ggtccgaccc 180 gatcttttga ctcagttggagcaagaagcc atcaacttct ttgctttgca tcactctctc 240 aaagacaaag cgggtccacctgacccgttt ggttacggta ctaaaaggat tggacccaat 300 ggtgaccttg gctggcttgagtacattctc cttaatgcta atctttgcct tgagtctcac 360 aaaaccaccg ccattttccggcacacccct gcaattttca gagaggcagt ggaagagtac 420 attaaagaga tgaagagaatgtcgagcaaa tttctggaaa tggtagagga agagctaaag 480 atagagccaa aggagaagctgagccgtttg gtgaaagtga aagaaagtga ttcgtgcctg 540 agaatgaacc attacccggagaaggaagag actccggtca aggaagagat tgggttcggt 600 gagcacactg atccacagttgatatcactg ctcagatcaa acgacacaga gggtttgcaa 660 atctgtgtca aagatggaacatgggttgat gttacacctg atcactcctc tttcttcgtt 720 cttgtcggag atactcttcaggtgatgaca aacggaagat tcaagagtgt gaaacataga 780 gtggtgacaa atacaaagaggtcaaggata tcgatgatct acttcgcagg tcctcctttg 840 agcgagaaga ttgcaccattatcatgcctt gtgccaaagc aagatgattg cctttataat 900 gagtttactt ggtctcaatacaagttatct gcttacaaaa ctaagcttgg tgactatagg 960 cttggtctct ttgagaaacgacctccattt tctctatcca atgtttga 1008 10 335 PRT Arabidopsis thaliana 10Met Val Ile Val Leu Gln Pro Ala Ser Phe Asp Ser Asn Leu Tyr Val 1 5 1015 Asn Pro Lys Cys Lys Pro Arg Pro Val Leu Ile Pro Val Ile Asp Leu 20 2530 Thr Asp Ser Asp Ala Lys Thr Gln Ile Val Lys Ala Cys Glu Glu Phe 35 4045 Gly Phe Phe Lys Val Ile Asn His Gly Val Arg Pro Asp Leu Leu Thr 50 5560 Gln Leu Glu Gln Glu Ala Ile Asn Phe Phe Ala Leu His His Ser Leu 65 7075 80 Lys Asp Lys Ala Gly Pro Pro Asp Pro Phe Gly Tyr Gly Thr Lys Arg 8590 95 Ile Gly Pro Asn Gly Asp Leu Gly Trp Leu Glu Tyr Ile Leu Leu Asn100 105 110 Ala Asn Leu Cys Leu Glu Ser His Lys Thr Thr Ala Ile Phe ArgHis 115 120 125 Thr Pro Ala Ile Phe Arg Glu Ala Val Glu Glu Tyr Ile LysGlu Met 130 135 140 Lys Arg Met Ser Ser Lys Phe Leu Glu Met Val Glu GluGlu Leu Lys 145 150 155 160 Ile Glu Pro Lys Glu Lys Leu Ser Arg Leu ValLys Val Lys Glu Ser 165 170 175 Asp Ser Cys Leu Arg Met Asn His Tyr ProGlu Lys Glu Glu Thr Pro 180 185 190 Val Lys Glu Glu Ile Gly Phe Gly GluHis Thr Asp Pro Gln Leu Ile 195 200 205 Ser Leu Leu Arg Ser Asn Asp ThrGlu Gly Leu Gln Ile Cys Val Lys 210 215 220 Asp Gly Thr Trp Val Asp ValThr Pro Asp His Ser Ser Phe Phe Val 225 230 235 240 Leu Val Gly Asp ThrLeu Gln Val Met Thr Asn Gly Arg Phe Lys Ser 245 250 255 Val Lys His ArgVal Val Thr Asn Thr Lys Arg Ser Arg Ile Ser Met 260 265 270 Ile Tyr PheAla Gly Pro Pro Leu Ser Glu Lys Ile Ala Pro Leu Ser 275 280 285 Cys LeuVal Pro Lys Gln Asp Asp Cys Leu Tyr Asn Glu Phe Thr Trp 290 295 300 SerGln Tyr Lys Leu Ser Ala Tyr Lys Thr Lys Leu Gly Asp Tyr Arg 305 310 315320 Leu Gly Leu Phe Glu Lys Arg Pro Pro Phe Ser Leu Ser Asn Val 325 330335 11 20 DNA Artificial Sequence Description of Artificial SequencePrimer 11 taatcactat ccaccatgtc 20 12 20 DNA Artificial SequenceDescription of Artificial Sequence Primer 12 tggagagagt cacccacgtt 20 1320 DNA Artificial Sequence Description of Artificial Sequence Primer 13ggttatgact aacgggaggt 20 14 20 DNA Artificial Sequence Description ofArtificial Sequence Primer 14 cttgtaagca gaagatttgt 20 15 30 DNAArtificial Sequence Description of Artificial Sequence Primer 15tgagctcaac catggttgtt ctgtctcagc 30 16 29 DNA Artificial SequenceDescription of Artificial Sequence Primer 16 tgagctctta atcagcagcagatttctgg 29

What is claimed is:
 1. A method of inhibiting plant growth comprising:(a) transforming a plant cell with a vector having a nucleic acid whichexpresses a plant polypeptide having gibberellin 2-oxidase enzymeactivity; wherein said polypeptide is expressed at a level sufficient toinhibit growth in a plant; and (b) growing a plant from said transformedplant cells.
 2. The method of claim 1, wherein said polypeptide is agibberellin 2-oxidase enzyme from Phaseolus or Arabidopsis.
 3. Themethod of claim 2, wherein said polypeptide is a gibberellin 2-oxidaseenzyme from Phaseolus coccineus or Arabidopsis thaliana.
 4. The methodof claim 1, wherein said nucleic acid comprises nucleotides 68 to 1063of SEQ ID NO:1.
 5. The method of claim 4, wherein said nucleic acidcomprises SEQ ID NO:1.
 6. The method of claim 1, wherein said nucleicacid encodes a polypeptide with an amino acid sequence consistingessentially of SEQ ID NO:2.
 7. The method of claim 1, wherein saidnucleic acid comprises nucleotides 41 to 1027 of SEQ ID NO:5.
 8. Themethod of claim 7, wherein said nucleic acid comprises SEQ ID NO:5. 9.The method of claim 1, wherein said nucleic acid encodes a polypeptidewith an amino acid sequence consisting essentially of SEQ ID NO:6. 10.The method of claim 1, wherein said nucleic acid comprises nucleotides109 to 1131 of SEQ ID NO:7.
 11. The method of claim 10, wherein saidnucleic acid comprises SEQ ID NO:7.
 12. The method of claim 1, whereinsaid nucleic acid encodes a polypeptide with an amino acid sequenceconsisting essentially of SEQ ID NO:8.
 13. The method of claim 1,wherein said nucleic acid comprises SEQ ID NO:9.
 14. The method of claim1, wherein said nucleic acid encodes a polypeptide with an amino acidsequence consisting essentially of SEQ ID NO:10.
 15. The method of claim1, wherein said nucleic acid comprises a coding sequence operativelylinked to a promoter.
 16. The method of claim 15, wherein said promoteris a constitutive promoter.
 17. The method of claim 15, wherein saidpromoter is specific for expression in a particular plant cell.
 18. Themethod of claim 1, wherein said expression of said polypeptide havingthe activity of a gibberellin 2-oxidase enzyme results in a reducedconcentration of bioactive gibberellins in said plant.
 19. The method ofclaim 1, wherein said polypeptide catalyses the 2β-oxidation of aC₁₉-gibberellin molecule to introduce a hydroxyl group at C-2.
 20. Themethod of claim 19, wherein said polypeptide further catalyses theoxidation of the hydroxyl group introduced at C-2 to yield the ketonederivative.
 21. The method of claim 1 wherein said inhibition of plantgrowth reduces bolting.
 22. An isolated nucleic acid comprisingnucleotides 68 to 1063 of SEQ ID NO:1.
 23. The nucleic acid of claim 22comprising SEQ ID NO:1.
 24. An isolated nucleic acid which encodes apolypeptide with an amino acid sequence consisting essentially of SEQ IDNO:2.